Designing materials with cues that direct stem cell differentiation into cartilage cells is a key step in developing tissue-engineering based strategie for craniofacial repair. By contributing to homogeneous differentiation and maintaining the desired differentiation state in vivo, these materials would increase the efficiency of differentiation and safety of cell-based therapies. The long-term goal is to establish general design rules for material properties that enhance or direct adult stem cell differentiation. To achieve this goal, the objective is to investigate chondrogenesis using a family of protein-based materials in which the biochemical and biophysical properties are decoupled and can be independently tuned. Specifically, we will incorporate sequences from resilin to confer the desired mechanical properties. We will also engineer protein-protein interactions within the material to modulate presentation of biochemical cues. Our central hypothesis is that material properties (i.e. ligand presentation and crosslinking density) can be used to modulate both mesenchymal stem cell (MSC) chondrogenesis and chondrocyte hypertrophy. In the first specific aim, we will investigate the effect of ligand presentation on cartilage differentiation. Through protein engineering, we will engineer materials that vary the binding strength of the ligand to the material and the valency of the ligand. The second specific aim will determine whether biochemical context modulates the optimal crosslinking density that maintains the cartilage phenotype and prevents hypertrophy. The innovation of this work is that we use the high level of structural control inherent in recombinant proteins to investigate materials parameters (e.g. binding strength and valency) that would not easily be achieved using other materials. The significance of the proposed research is that these protein-based materials will be used in establishing general design principles for cartilage-promoting materials.